engleski

Genetic duplications as a side-effect of targeted gene replacement in yeast

Background/aims: Introduction of linear, non replicative DNA fragments is widely used for the replacement of homologous sequences present in the yeast genome. This procedure is very useful for the construction of different yeast strains but is also studied as a general model for targeted genetic manipulations by the "ends-out" recombination. It is generally assumed that, in contrast to higher eukaryotes, transformation of the yeast Saccharomyces cerevisiae with non-replicative DNA occurs almost exclusively by homologous recombination. However, we found out that the ends-out transformation may result in different genetic alterations in the yeast genome and here we performed a systematic study of genetic events that may arise as side-effects of gene replacement. Method: Different alleles of the URA3 gene were present both on the transforming DNA fragment and in the yeast genome. The spectra of transformation evens were determined by Southern blot hybridization. Results: Depending on the experimental system and transformation method, targeted gene replacement ranged from only 60 % to 98%. Additionally, molecular analysis of transformants revealed different aberrant recombination events that included illegitimate integration of transforming DNA, tandem integrations, gene or whole chromosome duplications and chromosome fusions. Conclusions: We show that the fidelity of gene targeting in yeast is mainly influenced by the nature of alleles involved in recombination. The aberrant events observed include tandem and ectopic duplications as well as more complex chromosomal rearrangements. We propose that the ends-out recombination involving endogenous DNA fragments may have an important role in the evolution of the yeast genome.

gene targeting; gene duplication; aneuploidy; homologous recombination; illegitimate recombination

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